Crown gall is a widespread neoplastic disease caused by the bacterial pathogen Agrobacterium tumefaciens. Most dicotyledons, some gymnosperms, and a few monocotyledons are susceptible to the disease. (DeCleene and DeLey, 1976). It is known that large plasmids in virulent Agrobacterium strains are essential for tumor formation in infected plants. Such plasmids are referred to as Ti or tumor-inducing plasmids. (Simpson, et al , 1983.)
It is not yet known exactly how the bacterial pathogen transforms wounded host plants so they produce crown gall tumors. However, it is known the Ti (and the Ri or root-inducing plasmids from Agrobacterium rhizogenes) contain a region of DNA called the vir or virulence region that must be functional if wild-type cells are to be transformed into tumor cells. It is also known the end result of such transformation is the integration of another plasmid portion, called the T-DNA or transfer-DNA, into the nuclear genome of the transformed cells. The vir gene region is not integrated into the plant genome. It is however presumed to code for substances necessary for T-DNA transfer or integration, or both. (Schell and Van Montagu, 1983.)
Although functional vir genes are essential for T-DNA transfer, the vir genes and the T-DNA do not have to be carried on the same Ti plasmid for transfer and integration of the T-DNA to occur. The vir genes and the T-DNA can be carried on separate plasmids contained within the same Agrobacterium (Schell and Van Montagu, 1983). In fact, the octopine T-DNA segment can be transferred to plant cells if it is carried by an Agrobacterium containing the vir genes from a nopaline Ti plasmid, a limited host range Ti plasmid or an Ri plasmid (Hoekema, et al., 1984).
Native T-DNA, as opposed to T-DNA that has been "engineered" by man or mutated by nature, usually contains specific DNA border sequence(s) that flank: (1) oncogenic or "onco" genes (necessary for tumor formation) and, (2) genes that code for specific metabolites known as opines. There are several types of opines, none of which is produced by normal, i.e. non-transformed, plant cells. The type of opine produced by the tumor tissue, as well as the pattern of its production, is determined by the type of Ti plasmid carried in the infecting Agrobacterium. (Bomhoff, et al., 1976.) Thus, on the basis of the opine made in the plant tumor cell, the Ti plasmids have been grouped into octopine, nopaline and agropine classes. (Ooms, et al., 1982). The opines, although synthesized by the tumor tissue, are catabolized by the infecting bacteria. (Bomhoff, et al., 1976.)
Crown gall tumor, whether grown in vivo or maintained in vitro on simple medium, does not differentiate into sexually mature plants. Instead the tumor grows in an undifferentiated form referred to as a callus. Unlike wild-type plant cells, transformed cells from callus do not require the addition of plant hormones (auxins and cytokinins) to the growth medium in order to grow in culture. Rather calli can be maintained in culture on a simple medium that is devoid of exogenous growth substances. Since the plant hormones are known to play key roles in plant cell proliferation and differentiation, this lack of differentiation in the crown gall tumors is believed due to a growth hormone imbalance caused by the transforming Ti plasmids. (Simpson, et al., 1983.)
The Ti plasmids are natural plant vectors because they can transfer prokaryotic T-DNA into the eukaryotic plant cell. In addition, foreign heterologous genes can be inserted into the T-DNA of a Ti plasmid and thus genetically engineered into the genome of a plant cell. As a result, current efforts to transfer foreign genes into plant cells are centered on the Ti plasmids. (deFramond, et al., 1983.)
Unfortunately the Ti plasmids are too large to allow foreign DNA to be inserted directly into the T-DNA region. (Since restriction sites unique to the T-DNA region do not exist on these large plasmids it is extraordinarily difficult to utilize a specific site for the insertion of the heterologous DNA.) As a result, smaller "engineered" Ti-type vectors having fewer restriction sites must be utilized.
Once the foreign DNA has been inserted into the T-DNA on these smaller vectors, the vectors can be used to transfer the engineered T-DNA into the plant cell genome. Alternatively, a binary plant vector system, based on the separation of the vir and T-DNA regions, can be used. The binary system utilizes the interaction of two plasmids, one containing the vir region and the other containing the T-DNA region on a wide-host-range replicon. (An Agrobacterium tumefaciens strain harboring both plasmids has a normal tumor-inducing capacity although neither plasmid is functional alone.) With this approach, the T-DNA on one plasmid can, because of its smaller size, be easily manipulated to contain heterologous DNA when a bacterium such as Escherichia coli is used as a host. Subsequent transfer of the engineered plasmid into an Agrobacterium tumefaciens strain harboring the plasmid with the vir region makes it possible to introduce the manipulated or engineered T-DNA into the plant cell genome. (Hoekema, et al., 1983a.)
Although the Ti plasmids offer great promise as plant gene vectors, a major obstacle to their generalized use has been the difficulty in regenerating whole plants from the transformed tumor cells. Most disclosed regeneration methods involve in vitro tissue culture techniques. See generally Hoekama, et al. (1983b.) Some of these methods utilize an initial in vivo infection step that is followed by in vitro regeneration of transformed cells obtained from the crown gall callus. Other methods utilize two in vitro techniques, one to infect plant cell protoplasts and the other to regenerate whole plants from the transformed protoplasts. However, since these methods rely on in vitro plant tissue culture techniques, they are time consuming and burdensome to carry out. In addition, regeneration from protoplasts or calli has only been shown to be possible for a small number of plants. (Simpson, et al., 1983.)
More useful methods for transformation and regeneration of plant cells are ones that utilize in vivo infection methods for transformation but eliminate the need for in vitro regeneration. Instead these methods rely on in vivo regeneration of whole plants from tumors. Unfortunately, attempts to develop such methods have not been very successful. As a result, the art to date contains only a few reported cases of successful regeneration of transformed plants from tumors.
At least one of the reported cases (DeGreve, et al., 1982) apparently involved a fortuitous deletion of the oncogenes carried on the Ti plasmids. (Absence of the oncogenes allowed the transformed plant cells to differentiate into whole phenotypically normal plants.) In another, prior inactivation of one of the oncogenes allowed the infected plants to regenerate. See Barton, et al. (1983). Unfortunately this and other methods that involve crippled or disabled oncogenes are especially unsatisfactory for generalized use for plant transformation and regeneration because oncogenes, though disarmed, remain in the plant. It is possible these genes could be activated at some future point in the lifetime of the transformed plant or of its progeny. Since activation of the "disarmed" oncogenes could cause undesirable phenotypic changes in the plant, their presence presents an undesirable risk to the plant breeder seeking improved varieties of crop plants. In addition, the low frequency and unpredictable nature of the deletion process makes it unsatisfactory as a general method for regenerating whole plants from tumors.
Thus there has been a failure of past efforts to provide an in vivo method for transforming and regenerating whole plants from tumors wherein the plants are transformed to contain foreign DNA but not disabled oncogenes.